WO2009080482A1 - Pharmaceutical compositions comprising octahedral metal (iii) polypyridyl complexes and their use in prevention and treatment of cancer - Google Patents

Pharmaceutical compositions comprising octahedral metal (iii) polypyridyl complexes and their use in prevention and treatment of cancer Download PDF

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WO2009080482A1
WO2009080482A1 PCT/EP2008/067021 EP2008067021W WO2009080482A1 WO 2009080482 A1 WO2009080482 A1 WO 2009080482A1 EP 2008067021 W EP2008067021 W EP 2008067021W WO 2009080482 A1 WO2009080482 A1 WO 2009080482A1
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dmso
complexes
mer
fac
rhci
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PCT/EP2008/067021
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Ronald Gust
Ingo Ott
William S. Sheldrick
Melanie Harlos
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Freie Universität Berlin
RUHR-UNIVERSITäT BOCHUM
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0033—Iridium compounds
    • C07F15/004—Iridium compounds without a metal-carbon linkage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0073—Rhodium compounds
    • C07F15/008—Rhodium compounds without a metal-carbon linkage

Definitions

  • compositions comprising octahedral metal (III) polypyridyl complexes and their use in prevention and treatment of cancer
  • the present invention relates to pharmaceutical compositions comprising octahedral trihalido metal (III) polypyridyl complexes as well as to new octahedral trihalido metal (III) polypyridyl complexes and their use as anticancer and antimetastatic agents.
  • compositions comprising one or more octahedral metal (III) polypyridyl complexes of general formula I
  • Me represents rhodium or iridium hal is a halogenide selected from the group consisting of chlorine, bromine, fluorine and iodine or a pseudohalogenide selected from the group consisting of SCN, NCO or N 3
  • sol is a solvent selected from the group consisting of DMSO, H 2 O, CH 3 OH, DMF and CH 3 CN
  • pp is a polypyridyl ligand selected from the group constisting of 2,2'-bipyridine (bpy), 1 ,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz) and benzo[i]dipyrido[3,2-a:2',3'- c]phenazine (dppn), optionally substituted with one or more of the substituents selected from the group constisting
  • compositions comprising the complexes or their isomers and the hydrates, solvates or salts of the complexes or the isomers as active substances. These active substances exhibit significant cytotoxic effects which are superior to those of known cytostatic metal containing drugs as for instance cisplatin.
  • sol in formula I preferably means DMSO or H 2 O.
  • Hal according to formula I means preferably chlorine or bromine and pseudohalogenide means preferably SCN.
  • the preferred transition metal of the complex is rhodium.
  • Rhodium complexes of formula I with dpq, dppz or dppn als polypyridyl ligand exhibit superior cytotoxic effects in cell cultures which are orders of magnitude stronger than for cisplatin (compare Tab. 1 ).
  • iridium complexes of formula I with dppz and dppn as polypyridyl ligand (compare Tab. 2).
  • the substituents -COOR, -OH, -CHO and -SO 3 H are especially preferred to improve the solubility of the compounds of the invention in aqueous solutions, if necessary.
  • the polypyridyl ligand bpy may be monosubstituted with CH 3 , OCH 3 , OEt, Ph, CHO, CN, COOH, NH 2 , NO 2 , OH, SO 3 H, Cl or Br in position 3, 4 or 5 or disubstituted in positions 3,3'; 4,4'; 5,5'; 4,6 or 3,5 with these substituents.
  • the polypyridyl ligand phen may be monosubstituted in positions 2, 3, 4 or 5 or disubstituted in positions 2,9; 3,8; 4,7 or 5,6 with CH 3 , OCH 3 , OEt, Ph, CHO, CN, COOH, NO 2 , OH, SO 3 H, Cl or Br.
  • the polypyridyl ligand dpq may bear one of substituents -COOR, -CHO, -CH 3 , halogenide, hydroxy, phenyl, -CN or -NH 2 in position 2 or 1 1 or two of these substituents in positions 2,9; 4,7; 1 1 ,12; 3,8.
  • Preferred dpq complexes are such with - CH 3 , -CHO Or -CH 2 OH in 2-position and in positions 1 1 and 12 -COOC 2 H 5 .
  • Especially preferred dpq compounds are such with -COOH disubstituted in 4, 4'-position or in 1 1 , 12-position or disubstituted with -CN in 1 1 , 12-position.
  • the polypyridyl ligand dppz may be disubstuted in 12, 13-position with -CH 3 , -CN, - NO 2 , halogenide or phenyl, in 2, 9-position with -NH 2 , -CH 3 , -COOH or chloride, in 1 1 , 14-position with phenyl and bromide, in 3, 8-position with chloride, in 3, 4-position with -CH 3 and in 4, 7-position with CH 3 or phenyl.
  • dppz complexes with -CF 3 in 2-position and hydroxy or halogenide in 4-position are also suitable.
  • Monosubstituted dppz complexes with halogenide in 2-position, hydroxy or methyl in position 1 1 or -COOH, -NH 2 , -NO 2 , halogenide or OCH 3 in 12-position can also be used according to the invention.
  • the polypyridyl ligand dppn may be substituted with alkyl (for instance - CH 3 or n-butyl) in 2, 9-position.
  • the complexes are hydrates or solvates, preferably of formula Ia
  • n O; 1 ; 1 ,5; 2 or 3.
  • the complexes are isomers, especially fac- and mer-isomers, and hydrates or solvates of the isomers, preferably of formula Ia.
  • Preferred fac and mer isomers of the invention are mer-RhCI 3 (DMSO)(pp), fac- RhCI 3 (DMSO)(Pp), mer-RhCI 3 (DMSO-H 2 O)(pp), fac-RhCI 3 (DMSO-H 2 O)(pp), fac- RhCI 3 (H 2 O)(Pp), mer-RhCI 3 (H 2 O)(pp), fac-lrCI 3 (DMSO)(pp), mer-lrCI 3 (DMSO)(pp), mer-lrCI 3 (DMSO)(pp), fac-
  • Especially preferred isomeric complexes of the pharmaceutical compositions of the invention are mer-RhCI 3 (DMSO)(bpy), mer-RhCI 3 (DMSO)(phen) H 2 O, mer-RhCI 3 (DMSO)(dpq), mer-RhCI 3 (DMSO)(dppz) 1 ,5H 2 O, mer-RhCI 3 (DMSO)(dppn), fac-[lrCI 3 (DMSO)(bpy)] 2H 2 O, fac-[lrCI 3 (DMSO)(phen) CH 3 OH H 2 O, fac-[lrCI 3 (DMSO)(dpq)] 3H 2 O, fac-[lrCI 3 (DMSO)(dppz)] 2H 2 O, fac-[lrCI 3 (DMSO)(dppn) 2CH 3 OH, fac-[lrBr 3 (H 2 O)(phen)].
  • compositions of the invention also include the pharmaceutically acceptable salts of the complexes, or any other compound which, upon administration to the human subject, is capable of providing (directly or indirectly) the therapeutically active agent.
  • Salts according to the invention which may be conveniently used in therapy include physiologically acceptable base salts, e.g. derived from an appropriate base, such as alkali metal (e.g. sodium) salts, alkaline earth metal (e.g. magnesium) salts or ammonium salts.
  • physiologically acceptable base salts e.g. derived from an appropriate base, such as alkali metal (e.g. sodium) salts, alkaline earth metal (e.g. magnesium) salts or ammonium salts.
  • the pharmaceutical compositions comprise one or more octahedral metal (III) polypyridyl complexes of formula I as active substance in an amount sufficient to exhibit a therapeutic effect.
  • the pharmaceutical compositions may also comprise conventional auxiliary substances, preferably carriers, adjuvants and/or vehicles.
  • said carriers can be fillers, extenders, binders, humectants, disintegrants, dissolution retarders, absorption enhancers, wetting agents, adsorbents, and/or lubricants.
  • the pharmaceutical compositions of the invention may be prepared as a gel, powder, tablet, sustained-release tablet, premix, emulsion, infusion formulation, drops, concentrate, granulate, syrup, pellet, bolus or capsule and/or used in this form.
  • the pharmaceutical composition of the present invention can be administered orally in any orally tolerable dosage form, including capsules, tablets and aqueous suspensions and solutions, without being restricted thereto.
  • carriers frequently used include microcrystalline cellulose, lactose and corn starch.
  • lubricants such as magnesium stearate can be added.
  • useful diluents such as lactose and dried corn starch are employed.
  • aqueous suspensions the active substance is combined with emulsifiers and suspending agents. Also, particular sweeteners and/or flavors and/or coloring agents can be added, if desired.
  • the complexes of formula I can also be present in micro-encapsulated form, optionally with one or more of the above-specified carriers.
  • ointments, pastes, creams and gels may include conventional carriers such as animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances.
  • the pharmaceutical composition according to the invention may include conventional carriers such as solvents, solubilizers, and emulsifiers such as water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils, especially cotton seed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol, glycerol formal, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty esters of sorbitan, or mixtures of these substances.
  • the solutions and emulsions may also be present in a sterile and blood- isotonic form.
  • suspensions may include conventional carriers such as liquid diluents, e.g. water, ethyl alcohol, propylene glycol, suspending agents, e.g. ethoxylated isostearyl alcohols, polyoxyethylene-sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances.
  • liquid diluents e.g. water, ethyl alcohol, propylene glycol
  • suspending agents e.g. ethoxylated isostearyl alcohols, polyoxyethylene-sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances.
  • compositions can be present in the form of a lyophilized sterile injectable formulation, e.g. as a sterile injectable aqueous solution or aqueous or oily suspension.
  • a lyophilized sterile injectable formulation e.g. as a sterile injectable aqueous solution or aqueous or oily suspension.
  • Such a suspension can also be formulated by means of methods known in the art, using suitable dispersing or wetting agents (such as Tween 80) and suspending agents.
  • the production of the pharmaceutical formulations specified above proceeds in a usual manner according to well-known methods, e.g. by mixing the active substance(s) with the carrier(s).
  • the metal complexes of formula I may be encapsulated in semi-solid nanoparticles which prototypes are liposomes which serve as delivery systems.
  • Liposomes are completely closed lipid bilayer membranes enclosing an aqueous volume.
  • Liposomes can be unilamellar vesicles (i.e. having a single membrane bilayer) or multilamellar vesicles (i.e. onion-like structures characterized by several membrane bilayers each of which is separated from the next one by an aqueous layer).
  • the production of liposomes from saturated and unsaturated lipids has been described in a large number of patents, as well as their use as delivery systems for drugs.
  • the metal complexes may be encapsulated therein in a per se known manner. Such liposomes are mostly made from phospholipids. Alternatively, the metal complexes according to the invention may also be encapsulated in alginates or other gel-like structures.
  • the complexes of formula I are incorporated in a pharmaceutical formulation at a concentration of 0.1 to 99.5, preferably 0.5 to 95, and more preferably 20 to 80 wt.-%. That is, the active substance is present in the above pharmaceutical formulations, e.g. tablets, pills, granulates and others, at a concentration of preferably 0.1 to 99.5 wt.-% of the overall mixture.
  • the complexes of formula I are useful for the manufacture of a pharmaceutical composition for prevention and treatment of cancer.
  • carcinomas include carcinomas and sarkomas including haematological neoplasms.
  • Cancer is invasive and tends to metastasise to new sites. It spreads directly into surrounding tissues and also may be disseminated through the lymphatic and circulatory systems. Thus, any type of cancer can be treated according to the invention.
  • carcinomas which can be treated with the compositions of the invention are carcinomas of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, rectum, and stomach, especially breast, colon or colorectal carcinomas and their metastases.
  • these complexes are useful for preventing and treating colon and breast cancer.
  • Sarkomas which can be treated or prevented by the compositions of the invention are for instance lymphoma and leukaemia. Lymphomas often originate in lymph nodes, presenting as an enlargement of the node (a tumor).
  • Leukaemia include, but are not limited to, acute myelogenous leukaemia (AML), chronic myelogenous leukaemia (CML) and juvenile myelomonocytic leukaemia (JML), acute lymphocytic leukaemia (ALL) and chronic lymphocytic leukaemia (CLL).
  • AML acute myelogenous leukaemia
  • CML chronic myelogenous leukaemia
  • JML juvenile myelomonocytic leukaemia
  • ALL acute lymphocytic leukaemia
  • CLL chronic lymphocytic leukaemia
  • the present invention provides a method for treating cancer comprising adminstering to a patient suffering from cancer a therapeutically effective and safe amount of metal complexes of general formula I.
  • “Therapeutically effective amount” means an amount effective to yield the desired therapeutic response. For example, an amount effective to delay the growth of a cancer, to shrink or not metastasize.
  • “safe amount” refers to the quantity of a component that does not cause undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • the present invention provides new octahedral metal (III) polypyridyl complexes of formula Ib
  • Me represents rhodium or iridium hal is a halogenide selected from the group consisting of chlorine, bromine, fluorine and iodine or a pseudohalogenide selected from the group consisting of SCN, NCO Or N 3
  • sol is a solvent selected from the group consisting of DMSO, DMF and CH 3 CN
  • pp is a polypyridyl ligand selected from the group constisting of 2,2'-bipyridine (bpy), 1 ,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz) and benzo[i]dipyrido[3,2-a:2',3'- c]phenazine (dppn), optionally substituted with one or more of the substituents selected from the group constisting of hydroxy, -COOR,
  • Object of the invention are the complexes or their isomers and the hydrates, solvates or salts of the complexes or their isomers.
  • sol in formula Ib preferably represents DMSO.
  • Hal in formula Ib means preferably chlorine or bromine.
  • the polypyridyl ligand represents preferably phen, dpq, dppz or dppn, preferably dpq, dppz or dppn.
  • the complexes of formula Ib are hydrates or solvates, preferably complexes being [Me(hal)3(sol)(pp)](CH 3 OH) m (H 2 O) n , wherein m is 0; 1 or 2 and n is 0; 1 ; 1 ,5; 2 or 3.
  • the complexes of formula Ib are the fac- or mer-isomers.
  • Especially preferred isomeric complexes are mer-RhCI 3 (DMSO)(pp), fac-RhCI 3 (DMSO)(pp), mer-RhCI 3 (DMSO-H 2 O)(pp), fac- RhCI 3 (DMSO-H 2 O)(Pp), fac-lrCI 3 (DMSO)(pp), mer-lrCI 3 (DMSO)(pp), and their hydrates or solvates.
  • the complexes mer-RhCI 3 (DMSO)(pp) may be prepared by reaction of mer,cis- RhCI 3 (DMSO- ⁇ S) 2 (DMSO- ⁇ O)[12-14] with the appropriate polypyridyl ligand (bpy, phen, dpq, dppz, dppn) in CH 3 OH/H 2 O solution at about 75°C. Crystals of the fac- RhCI 3 (DMSO)(Pp) isomers may be grown over a period of about 7 days by slow evaporation of water/methanol solutions of the corresponding mer-isomers.
  • the complexes fac-lrCI 3 (DMSO)(pp) may be prepared by stepwise reaction of IrCI 3 3H 2 O with equimolar quantities of the appropriate polypyridyl ligand and DMSO in methanol solution in the dark. Crystals of the corresponding mer-lrCI 3 (DMSO)(pp) isomers may be obtained by slow evaporation of water/methanol solutions of the corresponding fac-isomers.
  • LSIMS spectra liquid secondary ion mass spectrometry
  • IrCIs H 2 O were purchased from Chempur, 1 ,10-phenanthroline (phen) from Acros, and
  • Anisotropic temperature factors were employed for the non-hydrogen atoms with the exception of the disordered water oxygen atom of RhI b and protons were included at geometrically calculated positions as riding atoms.
  • RhI b The molecular structure of RhI b as established by X-ray structural analysis is depicted in Fig. 3.
  • Example 1 Synthesis of rhodium (III) polypyridyl complexes of type mer- and fac-RhCI 3 (DMSO)(pp)
  • RhIa The general procedure is described below for RhIa.
  • Rh3a mer-[RhCI 3 (DMSO)(dpq)] Rh3a.
  • Rh1 dipyrido[3,2-/:2',3'- /7]quinoxaline (104.7 mg, 0.45 mmol). Yield: 76 %.
  • LSIMS m/z (%) 483(28) [M-Cl] + , 405(9) [M-CI-DMSO] + , 370(100) [M-2CI-DMSO] + , 335(83) [M-3CI-DMSO] + .
  • RMa - Rh5a The mer-isomers of RMa - Rh5a are stable in chloroform solution, those of RhIa and Rh2a isomerize rapidly to a mixture of fac- and mer-isomers in DMSO.
  • UVA/is and CD studies of the interaction of RMa - Rh5a with calf thymus DNA are in accordance with an absence of intercalation and time dependent 1 H NMR indicates that the complexes do not react with the guanine N7 atom of 5'-GMP 2" .
  • Example 2 Cytotoxicity Measurements of mer-RhCI 3 (DMSO)(pp) complexes MCF-7 breast cancer and HT-29 human colon carcinoma cells were maintained in 10 % (v/v) fetal calf serum containing cell culture medium (minimum essential eagle supplemented with 2.2 g NaHCC>3, 110 mg/L sodium pyruvate and 50 mg/L gentamicin sulfate adjusted to pH 7.4) at 37°C/5 % CO 2 and passaged twice a week according to standard procedures.
  • the antiproliferative effects of RMa - Rh5a were determined by an established procedure [15].
  • Cells were suspended in cell culture medium (MCF-7: 10000 cells/mL, HT-29: 2850 cells/mL), and 100 ⁇ l_ aliquots thereof were plated in 96 well plates and incubated at 37°C/5 % CO 2 for 72 h (MCF-7) or 48 h (HT-29).
  • Stock solutions of the compounds in DMSO were freshly prepared and diluted with cell culture medium to the desired concentrations (final DMSO concentration: 0.1 % v/v). The medium in the plates was replaced with the medium containing the compounds in graded concentrations (six replicates).
  • Rh2a phen 0.40(0.06) 0.19(0.05)
  • Rh3a - Rh5a are extremely potent cytotoxic agents with IC 5 O values in the range 0.069 - 0.079 ⁇ M, that are some two orders of magnitude lower than for cisplatin.
  • Example 3 Activity of mer-[RhCl3(DMSO)(pp)] complexes towards lymphoma and leukemia cells
  • Rh3a and Rh4a The inhibition of cell proliferation by complexes Rh3a and Rh4a, was also evaluated in vitro in BJAB cells (Burkitt-like lymphoma cells). After an incubation period of 24 h, the viability and cell count were measured with a CASY@CellCounter and Analyser System, with the settings specifically defined for the requirements of the employed cells. The dose dependent decreasing of cell proliferation is depicted for the highly potent rhodium(lll) complexes in Figure 9. IDgg values for these complexes are listed in Table 3. It is apparent that the meridional complexes Rh3a and Rh4a are effective at low micromolecular concentrations in inhibiting cell proliferation for the lymphoma cells
  • the number of viable cells N are given in units of 10 mL " with values given as the % of control values ⁇ the estimated standard deviation.
  • FIG. 10 depicts the % values of viable cells on the basis of the LDH release established for BJAB cells after 3 h incubation with different concentrations of Rh3a and Rh4a.
  • Apoptosis in contrast to unspecific necrosis, requires a controlled and regulated mechanism leading to cell death.
  • DNA fragmentation (hypoploidy) is considered to be a typical effect of apoptotic cell death and, therefore, the induction of apoptosis for Rh3a and Rh4a was quantified by flow cytometric measurements of the DNA fragments after incubating lymphoma cells (BJAB) and NALM-6 cells 72 h with the complexes.
  • BJAB lymphoma cells
  • NALM-6 cells 72 h The numbers of apoptotic NALM-6 cells for different concentrations of Rh3a and Rh4a are illustrated in Figure 1 1 a. This depicts the DNA fragmentation (hypoploidy) after treatment for 72 h.
  • Acute lymphoblastic leukemia is the most common malignant disease in childhood.
  • Rh3a and Rh4a were incubated with leukemia cells taken from a patient with relapsed childhood ALL.
  • the isolated primary lymphoblasts were treated with Rh3a and Rh4a at the ID 50 concentrations established for BJAB cells and with the cytostatic drugs daunorubicin, doxorubicin and vinchristine.
  • complexes Rh3a and Rh4a appear to exhibit superior apoptosis induction in comparison to these standard drugs for the treatment of childhood ALL.
  • Rh3a and Rh4a trigger the mitochondrial pathway of apoptosis.
  • dose dependent loss of the mitochondrial membrane potential was observed for BJAB cells after 48 h of incubation with the meridional rhodium(lll) compounds.
  • the mitochondrial permeability was quantified by flow cytometric determination of the cells with decreased fluorescence, i.e. with mitochondria displaying a lower membrane potential. Values of the mitochondrial permeability transition are given as percentages of cells with low ⁇ m .
  • BJAB burkitt-like lymphoma
  • NALM-6 human B cell precursor leukemia
  • Patients Primary lymphoblasts were obtained by bone marrow aspiration of patients with relapsed acute lymphoblastic leukemia (ALL). The diagnosis was established by immunophenotyping of leukemia cells according to Bene et * Lymphoblasts and mononuclear cells were separated by centrifugation over Biocoll (Biochrom KG, Berlin, Germany). After separation, the percentage of leukemia cells was above 95%.
  • ALL acute lymphoblastic leukemia
  • the leukemia cells were immediately seeded at a density of 3-10 5 cells mL in RPMI 1640 completed cell culture medium and incubated for 60 h with daunorubicin, doxorubicin and vincristine, as well as with complexes Rh3a and Rh4a at concentrations of their LD ⁇ Q values in BJAB cells.
  • the use of the cells is in accordance with the ethical standards of the responsible committee on human experimentation and the Helsinki Declaration as revised in 2000. It is also in accordance with the positive vote of the ethics committee from 14.12.2000 for the ALL-REZ-BFM-study in 2002. Informed signed consent was obtained from either the patient or from their next of kin.
  • Cytotoxicity measurements Cytotoxicity of Rh3a and Rh4a towards BJAB cells was measured by release of lactate dehydrogenase (LDH) as described previously. ⁇ ⁇ After incubation with different concentrations of the complexes for 1 h or 3 h at 37°C, LDH activity released by BJAB cells was measured in the cell culture supernatants using the Cytotoxicity Detection Kit from Boehringer Mannheim ® (Mannheim, Germany). The supernatants were centrifuged at 1500 rpm for 5 min.
  • LDH lactate dehydrogenase
  • Cell-free supernatants (20 ⁇ L) were diluted with phosphate-buffered saline (PBS, 80 ⁇ L) and a reaction mixture containing 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride (INT), sodium lactate, oxidised nicotinamide adenine dinucleotide (NAD + ) and diaphorase (100 ⁇ L) was added. Time-dependent formation of the reaction product was the quantified photometrically at 490 nm. The maximum amount of LDH activity released by the cells was determined by lysis of the cells by using 0.1% Triton X-100 in culture medium and set as 100% cell death.
  • PBS phosphate-buffered saline
  • INT 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride
  • NAD + oxidised nicotinamide a
  • Cell viability was determined by using the CASY ® Cell Counter + Analyzer System from Innovatis (Bielefeld, Germany). Settings were specifically defined for the requirements of the used cells. With this system, the cell concentration can be analyzed simultaneously in three different size ranges: cell debris, dead cells, and viable cells. Cells were seeded at a density of 1 -10 5 cells/ml and treated with different concentrations of Rh3a and Rh4a; non-treated cells served as controls. After a 24 h incubation period at 37°C, cells were resuspended properly and 100 ⁇ l_ of each well was diluted in 10 ml. CASYton (ready-to-use isotonic saline solution) for an immediate automated count of the cells.
  • CASYton ready-to-use isotonic saline solution
  • Apoptotic cell death was determined by a modified cell cycle-analysis, which detects DNA fragmentation at the single cell level as described previously. ⁇ ⁇ Cells were seeded at a density of 1 -10 5 cells mL and treated with different concentrations of Rh3a and Rh4a. After a 72 h incubation period at 37°C, cells were collected by centrifugation at 1500 rpm for 5 min, washed with PBS at 4 0 C and fixed in PBS/2% (v/v) formaldehyde on ice for 30 min. After fixation, cells were pelleted, incubated with ethanol/PBS (2:1 , v/v) for 15 min, pelleted and
  • Example 4 Synthesis of iridium (III) polypyridyl complexes of the type fac- and mer-lrCI 3 (DMSO)(pp)
  • the general procedure is described below for liia.
  • a Bruker DRX 400 was employed to record 1 H and 13 C NMR spectra with chemical shifts reported as ⁇ values relative to the signal of the deuterated solvent.
  • the fac isomers of liia - Ir5a are stable in light-protected CD 2 CI 2 solution but, with the exception of Ir5a, isomerize rapidly to a mixture of the fac and mer isomers in the presence of light.
  • CD 3 OD are stable under such conditions.
  • Ir2b was, however, isolated by slow evaporation of an H2O/CH3OH solution of Ir2a and characterized by X-ray structural analysis. UVA/is and CD studies of the interaction of IrIa - Ir5a with calf thymus DNA are in accordance with an absence of intercalation
  • HT-29 and MCF-7 cells were grown until at least 70 %
  • IrIa bpy > 100 > 100 n.d. n.d. 0.0 0.0
  • cytotoxity results are also depicted in Fig. 8 and indicate that, with the exception of the bpy complex Ilia, the compounds of the typ fac-lrCI 3 (DMSO)(pp) are potent cytotoxic agents, in particular the dppz and dppn complexes Ir4a and Ir5a.
  • DMSO fac-lrCI 3
  • Figure 3 Molecular structure of fac-[RhCI 3 (DMSO- ⁇ S)(bpy)] Rh1 b
  • Figure 4 IC 50 values for complexes RMa - Rh5a against the human cell lines MCF- 7 (breast cancer) and HT-29 (colon cancer)
  • Figure 8 IC 5 O values for the complexes Ir2a - Ir5a against the human cell lines MCF-7 (breast cancer) and HT-29 (colon cancer)
  • FIG. 10 BJAB cell viability values for complexes Rh3a and Rh4a. The cell viability was determined using the LDH release assay after an incubation period of 1 h.

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Abstract

The present invention relates to pharmaceutical compositions comprising octahedral trihalido metal (III) polypyridyl complexes as well as to new octahedral trihalido metal (III) polypyridyl complexes and their use as anticancer and antimetastatic agents.

Description

Pharmaceutical compositions comprising octahedral metal (III) polypyridyl complexes and their use in prevention and treatment of cancer
The present invention relates to pharmaceutical compositions comprising octahedral trihalido metal (III) polypyridyl complexes as well as to new octahedral trihalido metal (III) polypyridyl complexes and their use as anticancer and antimetastatic agents.
The trichloridorhodium (III) complexes mer,c/s-[RhCl3(DMSO-κS)(lm)2] (DMSO = (CHs)2SO, Im = imidazole) and mer,c/s-[RhCI3(DMSO-κS) 2(L)] (L = Im, NH3) have been studied by Mestroni et al. in 1998 [1] who established cytotoxic activity (IC50 = 1.5 ± 0.4, 0.4 ± 0.2, 9 μM) for the latter amine complex towards the human cell lines A 2780 (ovarian carcinoma), LoVo (colon carcinoma) and CaIu (lung carcinoma). Replacing ammonia by imidazole leads to an increase in the IC50 values by about an order of magnitude and mer,c/s-[RhCl3(DMSO-κS)(lm)2 is essentially inactive against A2780 and CaIu (IC50 > 200 μM) and only moderately active towards the colon carcinoma cell line LoVo (IC5O = 40 ± 15 μM). Activity against mouse P 388 leukemia has, however, been reported for the analogous compound mer,c/'s-[RhCl3(DMSO- κS)(py)2] (py = pyridine)[2]. The 2,2':6',2"-terpyridine (tpy) complexes mer-[RhCI3(tpy)] and [Rh(lm)(tpy)2]CI-3H2O also exhibit cytotoxicity [3], as does the compound fac- [RhCI3(9-[ane]-NS2)] (9-[ane]-NS2 = 1-aza-4,7-dithiacyclononane)[4].
Cytotoxic activity towards the human cell lines MCF-7 (breast cancer) and HT-29 (colon cancer) (IC50 = 2.3(0.4) and 7.4(0.9)μM) has also been found for the iridium (III) complex [(η5-C5Me5)lrCI(dppz)](CF3S03) (dppz = dipyrido[3,2-a:2',3'-c]phenazine)
(C5Me5 = pentamethylcyclopentadienyl) [5, 6]. However, in general, iridium complexes are even more inert than their Rh(III) analogues and [lmH][Yrans-lrCl4(lm)2], [ImH][^nS-IrCI4(DMSO)(Im)] (Im = imidazole) and [(DMSO)2H] [frans-lrCI4(DMSO)2] have all been found to be biologically inactive [7, 8]. These findings are in accordance with the general tenet, that the lack of reactivity of many Ir(III) complexes will correlate with an effective absence of cytotoxic effects on tumour cell lines, even at relatively high concentrations. The efficacy of available treatments on many cancer types is limited, and new, improved forms of treatment showing clinical benefit are needed. It is an object of the present invention to provide alternative pharmaceuticals compositions and compounds having potential to prevent and treat cancer and its metastases.
The problem of the invention is solved by the provision of the embodiments as defined in the claims of the present invention. It has been found that pharmaceutical compositions comprising one or more octahedral metal (III) polypyridyl complexes of general formula I
Me(hal)3(sol)(pp) (I)
where
Me represents rhodium or iridium hal is a halogenide selected from the group consisting of chlorine, bromine, fluorine and iodine or a pseudohalogenide selected from the group consisting of SCN, NCO or N3, sol is a solvent selected from the group consisting of DMSO, H2O, CH3OH, DMF and CH3CN and pp is a polypyridyl ligand selected from the group constisting of 2,2'-bipyridine (bpy), 1 ,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz) and benzo[i]dipyrido[3,2-a:2',3'- c]phenazine (dppn), optionally substituted with one or more of the substituents selected from the group constisting of hydroxy, -COOR, -SO3H, -CHO, -CH3, - CF3, -OCH3, -OC2H5, -NO2, -CN, -NH2, phenyl and halogenide, wherein R = H, - CH3 Or -C2H5,
and their physiologically tolerated isomers, and hydrates, solvates and salts thereof are useful cytotoxic agents for prevention and treatment of cancer and its metastases. Object of the invention are pharmaceutical compositions comprising the complexes or their isomers and the hydrates, solvates or salts of the complexes or the isomers as active substances. These active substances exhibit significant cytotoxic effects which are superior to those of known cytostatic metal containing drugs as for instance cisplatin.
According to the invention, sol in formula I preferably means DMSO or H2O.
Hal according to formula I means preferably chlorine or bromine and pseudohalogenide means preferably SCN.
According to the invention the preferred transition metal of the complex is rhodium.
With regard to the polypyridyl ligands of the complexes of the invention phen, dpq, dppz or dppn are preferred and dpq, dppz or dppn are especially preferred. Rhodium complexes of formula I with dpq, dppz or dppn als polypyridyl ligand exhibit superior cytotoxic effects in cell cultures which are orders of magnitude stronger than for cisplatin (compare Tab. 1 ). The same applies to iridium complexes of formula I with dppz and dppn as polypyridyl ligand (compare Tab. 2).
Optionally the polypyridyl ligands may be substituted with one or more of the substituents selected from the group consisting of one or more of the substituents selected from the group consisting of hydroxy, -COOR, -SO3H, -CHO, -CH3, -CF3, - OCH3, -OC2H5, -NO2, -CN, -NH2, phenyl and halogenide, wherein R = H, -CH3 or -C2H5. The substituents -COOR, -OH, -CHO and -SO3H are especially preferred to improve the solubility of the compounds of the invention in aqueous solutions, if necessary.
Particularly, the polypyridyl ligand bpy may be monosubstituted with CH3, OCH3, OEt, Ph, CHO, CN, COOH, NH2, NO2, OH, SO3H, Cl or Br in position 3, 4 or 5 or disubstituted in positions 3,3'; 4,4'; 5,5'; 4,6 or 3,5 with these substituents. The polypyridyl ligand phen may be monosubstituted in positions 2, 3, 4 or 5 or disubstituted in positions 2,9; 3,8; 4,7 or 5,6 with CH3, OCH3, OEt, Ph, CHO, CN, COOH, NO2, OH, SO3H, Cl or Br.
The polypyridyl ligand dpq may bear one of substituents -COOR, -CHO, -CH3, halogenide, hydroxy, phenyl, -CN or -NH2 in position 2 or 1 1 or two of these substituents in positions 2,9; 4,7; 1 1 ,12; 3,8. Preferred dpq complexes are such with - CH3, -CHO Or -CH2OH in 2-position and in positions 1 1 and 12 -COOC2H5. Especially preferred dpq compounds are such with -COOH disubstituted in 4, 4'-position or in 1 1 , 12-position or disubstituted with -CN in 1 1 , 12-position.
The polypyridyl ligand dppz may be disubstuted in 12, 13-position with -CH3, -CN, - NO2, halogenide or phenyl, in 2, 9-position with -NH2, -CH3, -COOH or chloride, in 1 1 , 14-position with phenyl and bromide, in 3, 8-position with chloride, in 3, 4-position with -CH3 and in 4, 7-position with CH3 or phenyl. According to the invention dppz complexes with -CF3 in 2-position and hydroxy or halogenide in 4-position are also suitable. Monosubstituted dppz complexes with halogenide in 2-position, hydroxy or methyl in position 1 1 or -COOH, -NH2, -NO2, halogenide or OCH3 in 12-position can also be used according to the invention.
Particularly, the polypyridyl ligand dppn may be substituted with alkyl (for instance - CH3 or n-butyl) in 2, 9-position.
In an embodiment of the invention the complexes are hydrates or solvates, preferably of formula Ia
[Me(hal)3(sol)(pp)] (CH30H)m (H2O)n, (Ia)
wherein m is O; 1 or 2 and n is O; 1 ; 1 ,5; 2 or 3.
In an other embodiment of the invention the complexes are isomers, especially fac- and mer-isomers, and hydrates or solvates of the isomers, preferably of formula Ia. Preferred fac and mer isomers of the invention are mer-RhCI3(DMSO)(pp), fac- RhCI3(DMSO)(Pp), mer-RhCI3(DMSO-H2O)(pp), fac-RhCI3(DMSO-H2O)(pp), fac- RhCI3(H2O)(Pp), mer-RhCI3(H2O)(pp), fac-lrCI3(DMSO)(pp), mer-lrCI3(DMSO)(pp), fac-
IrBr3(H2O)(Pp), mer-lrBr3(H2O)(pp), fac-lrCI3(H2O)(pp), mer-lrCI3(H2O)(pp).
Especially preferred isomeric complexes of the pharmaceutical compositions of the invention are mer-RhCI3(DMSO)(bpy), mer-RhCI3(DMSO)(phen) H2O, mer-RhCI3(DMSO)(dpq), mer-RhCI3(DMSO)(dppz) 1 ,5H2O, mer-RhCI3(DMSO)(dppn), fac-[lrCI3(DMSO)(bpy)] 2H2O, fac-[lrCI3(DMSO)(phen) CH3OH H2O, fac-[lrCI3(DMSO)(dpq)] 3H2O, fac-[lrCI3(DMSO)(dppz)] 2H2O, fac-[lrCI3(DMSO)(dppn) 2CH3OH, fac-[lrBr3(H2O)(phen)].
The above mentioned compositions of the invention also include the pharmaceutically acceptable salts of the complexes, or any other compound which, upon administration to the human subject, is capable of providing (directly or indirectly) the therapeutically active agent.
Salts according to the invention which may be conveniently used in therapy include physiologically acceptable base salts, e.g. derived from an appropriate base, such as alkali metal (e.g. sodium) salts, alkaline earth metal (e.g. magnesium) salts or ammonium salts.
According to the present invention the pharmaceutical compositions comprise one or more octahedral metal (III) polypyridyl complexes of formula I as active substance in an amount sufficient to exhibit a therapeutic effect.
The pharmaceutical compositions may also comprise conventional auxiliary substances, preferably carriers, adjuvants and/or vehicles. For example, said carriers can be fillers, extenders, binders, humectants, disintegrants, dissolution retarders, absorption enhancers, wetting agents, adsorbents, and/or lubricants. The pharmaceutical compositions of the invention may be prepared as a gel, powder, tablet, sustained-release tablet, premix, emulsion, infusion formulation, drops, concentrate, granulate, syrup, pellet, bolus or capsule and/or used in this form.
For example, the pharmaceutical composition of the present invention can be administered orally in any orally tolerable dosage form, including capsules, tablets and aqueous suspensions and solutions, without being restricted thereto. In case of tablets for oral application, carriers frequently used include microcrystalline cellulose, lactose and corn starch. Typically, lubricants such as magnesium stearate can be added. For oral administration in the form of capsules, useful diluents such as lactose and dried corn starch are employed. In oral administration of aqueous suspensions the active substance is combined with emulsifiers and suspending agents. Also, particular sweeteners and/or flavors and/or coloring agents can be added, if desired.
The complexes of formula I can also be present in micro-encapsulated form, optionally with one or more of the above-specified carriers.
In addition to the complexes of formula I as active substance(s), ointments, pastes, creams and gels may include conventional carriers such as animal and vegetable fats, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide or mixtures of these substances.
If the pharmaceutical composition according to the invention is provided as solution or emulsion it may include conventional carriers such as solvents, solubilizers, and emulsifiers such as water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethylformamide, oils, especially cotton seed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol, glycerol formal, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty esters of sorbitan, or mixtures of these substances. For parenteral application, the solutions and emulsions may also be present in a sterile and blood- isotonic form.
In addition to the complexes of formula I as active substance(s), suspensions may include conventional carriers such as liquid diluents, e.g. water, ethyl alcohol, propylene glycol, suspending agents, e.g. ethoxylated isostearyl alcohols, polyoxyethylene-sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, and tragacanth, or mixtures of these substances.
The pharmaceutical compositions can be present in the form of a lyophilized sterile injectable formulation, e.g. as a sterile injectable aqueous solution or aqueous or oily suspension. Such a suspension can also be formulated by means of methods known in the art, using suitable dispersing or wetting agents (such as Tween 80) and suspending agents.
The production of the pharmaceutical formulations specified above proceeds in a usual manner according to well-known methods, e.g. by mixing the active substance(s) with the carrier(s).
For administration, the metal complexes of formula I may be encapsulated in semi-solid nanoparticles which prototypes are liposomes which serve as delivery systems. Liposomes are completely closed lipid bilayer membranes enclosing an aqueous volume. Liposomes can be unilamellar vesicles (i.e. having a single membrane bilayer) or multilamellar vesicles (i.e. onion-like structures characterized by several membrane bilayers each of which is separated from the next one by an aqueous layer). The production of liposomes from saturated and unsaturated lipids has been described in a large number of patents, as well as their use as delivery systems for drugs. The metal complexes may be encapsulated therein in a per se known manner. Such liposomes are mostly made from phospholipids. Alternatively, the metal complexes according to the invention may also be encapsulated in alginates or other gel-like structures.
According to the invention the complexes of formula I are incorporated in a pharmaceutical formulation at a concentration of 0.1 to 99.5, preferably 0.5 to 95, and more preferably 20 to 80 wt.-%. That is, the active substance is present in the above pharmaceutical formulations, e.g. tablets, pills, granulates and others, at a concentration of preferably 0.1 to 99.5 wt.-% of the overall mixture. According to a further embodiment of the invention the complexes of formula I are useful for the manufacture of a pharmaceutical composition for prevention and treatment of cancer.
According to the present invention "cancer" includes malignant neoplasms that can be divided into two categories: carcinomas and sarkomas including haematological neoplasms.
Cancer is invasive and tends to metastasise to new sites. It spreads directly into surrounding tissues and also may be disseminated through the lymphatic and circulatory systems. Thus, any type of cancer can be treated according to the invention.
Among the carcinomas which can be treated with the compositions of the invention are carcinomas of the bladder, breast, colon, kidney, liver, lung, ovary, pancreas, rectum, and stomach, especially breast, colon or colorectal carcinomas and their metastases. In a preferred embodiment of the invention these complexes are useful for preventing and treating colon and breast cancer.
Sarkomas which can be treated or prevented by the compositions of the invention are for instance lymphoma and leukaemia. Lymphomas often originate in lymph nodes, presenting as an enlargement of the node (a tumor). Leukaemia include, but are not limited to, acute myelogenous leukaemia (AML), chronic myelogenous leukaemia (CML) and juvenile myelomonocytic leukaemia (JML), acute lymphocytic leukaemia (ALL) and chronic lymphocytic leukaemia (CLL). In a preferred embodiment of the invention these complexes of the compositions are useful for preventing and treating lymphoma and ALL.
According to a further embodiment the present invention provides a method for treating cancer comprising adminstering to a patient suffering from cancer a therapeutically effective and safe amount of metal complexes of general formula I.
"Therapeutically effective amount" means an amount effective to yield the desired therapeutic response. For example, an amount effective to delay the growth of a cancer, to shrink or not metastasize. "Safe amount" refers to the quantity of a component that does not cause undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
According to another embodiment the present invention provides new octahedral metal (III) polypyridyl complexes of formula Ib
Me(hal)3(sol)(pp) (Ib)
where
Me represents rhodium or iridium hal is a halogenide selected from the group consisting of chlorine, bromine, fluorine and iodine or a pseudohalogenide selected from the group consisting of SCN, NCO Or N3, sol is a solvent selected from the group consisting of DMSO, DMF and CH3CN and pp is a polypyridyl ligand selected from the group constisting of 2,2'-bipyridine (bpy), 1 ,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz) and benzo[i]dipyrido[3,2-a:2',3'- c]phenazine (dppn), optionally substituted with one or more of the substituents selected from the group constisting of hydroxy, -COOR, -SO3H, -CHO, -CH3, - CF3, -OCH3, -OC2H5, -NO2, -CN, -NH2, phenyl and halogenide, wherein R = H, - CH3 Or -C2H5, and their physiologically tolerated isomers, and hydrates, solvates or salts thereof.
Object of the invention are the complexes or their isomers and the hydrates, solvates or salts of the complexes or their isomers. According to the invention, sol in formula Ib preferably represents DMSO. Hal in formula Ib means preferably chlorine or bromine.
The polypyridyl ligand represents preferably phen, dpq, dppz or dppn, preferably dpq, dppz or dppn. In an embodiment of the invention the complexes of formula Ib are hydrates or solvates, preferably complexes being [Me(hal)3(sol)(pp)](CH3OH)m (H2O)n, wherein m is 0; 1 or 2 and n is 0; 1 ; 1 ,5; 2 or 3.
In another embodiment of the invention the complexes of formula Ib are the fac- or mer-isomers. Especially preferred isomeric complexes are mer-RhCI3(DMSO)(pp), fac-RhCI3(DMSO)(pp), mer-RhCI3(DMSO-H2O)(pp), fac- RhCI3(DMSO-H2O)(Pp), fac-lrCI3(DMSO)(pp), mer-lrCI3(DMSO)(pp), and their hydrates or solvates.
The general preparation procedure is described in the Examples of the present invention. Complexes not mentioned in the examples can be prepared in analogy using the corresponding starting compounds.
The complexes mer-RhCI3(DMSO)(pp) may be prepared by reaction of mer,cis- RhCI3(DMSO-κS)2(DMSO-κO)[12-14] with the appropriate polypyridyl ligand (bpy, phen, dpq, dppz, dppn) in CH3OH/H2O solution at about 75°C. Crystals of the fac- RhCI3(DMSO)(Pp) isomers may be grown over a period of about 7 days by slow evaporation of water/methanol solutions of the corresponding mer-isomers.
The complexes fac-lrCI3(DMSO)(pp) may be prepared by stepwise reaction of IrCI33H2O with equimolar quantities of the appropriate polypyridyl ligand and DMSO in methanol solution in the dark. Crystals of the corresponding mer-lrCI3(DMSO)(pp) isomers may be obtained by slow evaporation of water/methanol solutions of the corresponding fac-isomers.
Without intending to be limiting, the invention will be explained in more detail with reference to the following examples.
Materials and Instrumentation
UVA/is spectra were recorded with an Analytik Jena SPECORD 200 spectrometer and CD spectra with a Jasco J-715 instrument in the range 220 - 500 nm for 1 :10 complex/[DNA] mixtures [complex = 20 μM, DNA concentration in M(nucleotide) = 200 μM] in a 10 mM phosphate buffer at pH 7.2. 1 % DMSO was added to assure solubility of RMa - Rh5a. LSIMS spectra (liquid secondary ion mass spectrometry) were registered for the mass range m/z < 3000 with a Fisons VG autospec employing a caesium ion gun (voltage 17 kV) and 3-nitrobenzyl alcohol as the liquid matrix. A
1 13
Bruker DRX 400 was employed for the registration of H and C NMR spectra with chemical shifts reported as δ values relative to the signal of tetramethylsilane. Atomic absorption spectrometric measurements were performed on a Vario 6 (Analytik Jena) and elemental analyses on a Vario EL (Elementar Analysensysteme). RhCI 3H O and
IrCIs H2O were purchased from Chempur, 1 ,10-phenanthroline (phen) from Acros, and
2,2'-bipyridine (bpy) and dimethylsulfoxide (DMSO) from J. T. Baker. The polypyridyl ligands dpq [9], dppz [10] und dppn [1 1] were prepared in accordance with literature procedures as was the starting compound mer,c/s-[RhCI3(DMS0-κS)2(DMS0-κ0)] [12] and the complexes [(η5-C5Me5)lrCI(dppz)](CF3S03) [5] and [(η5- C5Me5)IrUMe2N)2CSKdPPn)](CF3SO3) [5].
X-ray Structural Analyses
Intensity data for fac-[RhCI3(DMSO-κS)(bpy)] H2O RhI b were collected using ω scans on a Siemens P4 diffractometer equipped with graphite-monochromated MoKa radiation (λ = 0.71073 A, 4° < 2Θ < 50°). The data were corrected semi-empirically for absorption (ψ scans) and the structures were solved by direct methods and refined by full-matrix least squares against F0 2 using SHELX97 (G. M. Sheldrick, SHELXS97 and SHELXL97, Gottingen, Germany, 1997). Anisotropic temperature factors were employed for the non-hydrogen atoms with the exception of the disordered water oxygen atom of RhI b and protons were included at geometrically calculated positions as riding atoms. The final R factors were Ri= 0.047 and 0.033 for I > 2σ (I) with wR2 = 0.1 19 and 0.083 for all independent reflections.
The molecular structure of RhI b as established by X-ray structural analysis is depicted in Fig. 3.
Example 1 : Synthesis of rhodium (III) polypyridyl complexes of type mer- and fac-RhCI3(DMSO)(pp) The rhodium(lll) complexes mer-RhCI (DMSO- S)(pp) Rh1a-Rh5a (pp = bpy, phen, dpq, dppz, dppn) were prepared by treatment of the precursor me/-,c/s-[RhCI3(DMSO- κS)2(DMS0-κ0)] [12-14] with an equivalent of the appropriate polypyridyl ligand in CH3OH/H2O solution (1/1 ). The general procedure is described below for RhIa.
mer-[RhCI3(DMSO)(bpy)] RhIa. mer,c/s-[RhCI3(DMS0-κ0)(DMS0-κS)2] (200 mg, 0.45 mmol) was dissolved in 10 ml. of a 1 :1 mixture of methanol and water. After addition of 2,2'-bipyridine (70.3 mg, 0.45 mmol), the reaction mixture was stirred for 2 h at 75 C and then left to stand at 4 C for a further 24 h. The resulting yellow precipitate was filtered off, treated with 5 ml. methanol and reprecipitated by addition of diethyl ether. The solid was filtered off, washed and dried in vacuo. Yield: 76 %. Anal.
(C12H14CI3N2ORhS) C, H, N: calcd. 32.49, 3.18, 6.32; found 32.30, 3.27, 6.36.
LSIMS: m/z (%) 407(100) [M-Cl]+, 372(37) [M-2CI]+. 1 H NMR (CDCI3): δ 3.71 (s, 6H,
CH3), 7.63 (dd, 1 H), 7.71 (dd, 1 H), 8.04 (dd, 1 H), 8.1 1 (d, 1 H) 8.14 (dd, 1 H), 8.16 (d, 1 H), 10.01 (d, 2H). Crystals of fac-[RhCI3(DMSO- S)(bpy)] H2O Rh1 b suitable for X- ray analysis (and characterized by X-ray analysis) were grown over a period of 7 days by slow evaporation of a solution of RhIa in water/methanol.
mer-[RhCI3(DMSO)(phen)] H2O Rh2a. Synthesis as for Rh1 with 1 ,10-phenanthroline (81.1 mg, 0.45 mmol). Yield: 74 %. Anal. (C14H1 6CI3N2O2RhS) C, H, N: calcd. 34.62,
3.32, 5.75; found 34.41 , 3.10, 5.99. LSIMS: m/z (%) 431 (100) [M-Cl]+, 391 (38) [M- 2Cl]+. 1 H NMR (CDCI3): δ 3.78 (s, 6H, CH3), 7.93 (dd, 1 H), 8.03 (dd, 1 H), 8.04 (dd, 1 H), 8.05 (d, 1 H) 8.50 (dd, 1 H), 8.58 (d, 1 H), 10.17 (d, 2H), 10.24 (d,1 H).
mer-[RhCI3(DMSO)(dpq)] Rh3a. Synthesis as for Rh1 with dipyrido[3,2-/:2',3'- /7]quinoxaline (104.7 mg, 0.45 mmol). Yield: 76 %. Anal. (C1 6H14CI3N4ORhS) C, H, N: calcd. 36.98, 2.72, 10.78; found 37.05, 2.96, 10.79. LSIMS: m/z (%) 483(28) [M-Cl]+, 405(9) [M-CI-DMSO]+, 370(100) [M-2CI-DMSO]+, 335(83) [M-3CI-DMSO]+. 1 H NMR (CDCI3): δ 3.84 (s, 6H, CH3), 8.13 (dd, 1 H), 8.23 (dd, 1 H), 9.19 (s, 2H), 9.70 (d, 1 H) 9.78 (d, 1 H), 10.33 (d, 1 H), 10.39 (d, 1 H).
mer-[RhCI3(DMSO)(dppz)]ϊ .5H2O Rh4a. Synthesis as for Rh1 with dipyrido[3,2- a:2',3'-c]phenazine (127.3 mg, 0.45 mmol). Yield: 71 %. Anal. (C2oHi 9CI3N4O2.5RhS)
C, H, N: calcd. 40.26, 9.37, 3.21 found 40.56, 8.97, 3.31. LSIMS: m/z (%) 533(19) [M- Cl]+, 458(9) [M-CI-DMSO]+, 420(65) [M-2CI-DMSO]+, 385(100). 1 H NMR (CDCI3): δ 3.75 (s, 6H, CH3) 8.00 (dd, 1 H), 8.02 (dd, 1 H), 8.1 1 (s, 1 H), 8.13 (s, 1 H) 8.38 (d, 1 H),
8.40 (d, 1 H), 9.76 (d, 1 H), 9.82 (d,1 H), 10.22 (d, 1 H), 10.28 (d, 1 H) ppm.
mer-[RhCI3(DMSO)(dppn)] Rh5a. Synthesis as for Rh1 with benzo[i]dipyrido[3,2- a:2',3'-c]phenazine (149.6 mg, 0.45 mmol). Yield: 68 %. Anal. (C24H1 8CI3N4ORhS) C,
H, N: calcd. 46.51 , 2.93, 9.04; found 46.42, 3.08, 8.96. LSIMS: m/z (%) 583(38) [M- Cl]+, 548(1 1 ) [M-2CI]+ 435(41 ) [M-3CI-DMSO]+. 1 H NMR (CDCI3): δ 3.80 (s, 6H, CH3) 7.68, 7.70 (2d, 2H), 8.07 (dd, 1 H), 8.16 (dd, 1 H), 8.25, 8.27 (2d, 2H) 9.06, 9.08 (2s, 2H), 9.80, 9.86 (2d, 2H), 10.25, 10.31 (2d, 2H).
The mer-isomers of RMa - Rh5a are stable in chloroform solution, those of RhIa and Rh2a isomerize rapidly to a mixture of fac- and mer-isomers in DMSO.
Both isomerization and rapid DMSO/H2O exchange are observed for aqueous solutions of the complexes. Hence, complexes of the type mer-RhCI3(DMSO-H2O)(pp) and/or fac-RhCI3(H2O)(pp) will probably be the biologically active species.
UVA/is and CD studies of the interaction of RMa - Rh5a with calf thymus DNA are in accordance with an absence of intercalation and time dependent 1H NMR indicates that the complexes do not react with the guanine N7 atom of 5'-GMP2".
The structures of the mer complexes RMa - Rh5a and of the fac complexes RM b - Rh2b are shown in Figures 1 and 2.
Example 2: Cytotoxicity Measurements of mer-RhCI3(DMSO)(pp) complexes MCF-7 breast cancer and HT-29 human colon carcinoma cells were maintained in 10 % (v/v) fetal calf serum containing cell culture medium (minimum essential eagle supplemented with 2.2 g NaHCC>3, 110 mg/L sodium pyruvate and 50 mg/L gentamicin sulfate adjusted to pH 7.4) at 37°C/5 % CO2 and passaged twice a week according to standard procedures. The antiproliferative effects of RMa - Rh5a were determined by an established procedure [15]. Cells were suspended in cell culture medium (MCF-7: 10000 cells/mL, HT-29: 2850 cells/mL), and 100 μl_ aliquots thereof were plated in 96 well plates and incubated at 37°C/5 % CO2 for 72 h (MCF-7) or 48 h (HT-29). Stock solutions of the compounds in DMSO were freshly prepared and diluted with cell culture medium to the desired concentrations (final DMSO concentration: 0.1 % v/v). The medium in the plates was replaced with the medium containing the compounds in graded concentrations (six replicates). After further incubation for 96 h (MCF-7) or 72 h (HT-29) the cell biomass was determined by crystal violet staining and the IC50 values were established as those concentrations causing 50 % inhibition of cell proliferation. Results were calculated from 2 - 3 independent experiments. The results are summarized in Tab. 1 and Fig. 4.
Table 1 IC50values (μM) for the complexes mer-[RhCI3(DMSO)(pp)] RMa - Rh5a
Complex pp MCF-7 IC50 HT-29 IC50
RMa bpy 4.0(0.5) 1.9(0.5)
Rh2a phen 0.40(0.06) 0.19(0.05)
Rh3a dpq 0.079(0.012) 0.069(0.021)
Rh4a dppz 0.095(0.020) 0.073(0.017)
Rh5a dppn 0.051(0.012) 0.070(0.008)
Cisplatin 2.0(0.3) 7.0(2.0) dppz 0.8(0.6) 1.8(0.2)
It is apparent for the complexes RMa - Rh3a that their IC5O values (bpy > phen > dpq) are strongly correlated to the surface area of the polypyridyl ligand. Whereas a dramatic improvement is observed for the smaller bpy, phen and dpq ligands of RMa - Rh3a, no further significant increase in cytotoxicity is apparent for the larger dppz and dppn ligands of Rh4a and Rh5a.
Complexes Rh3a - Rh5a are extremely potent cytotoxic agents with IC5O values in the range 0.069 - 0.079 μM, that are some two orders of magnitude lower than for cisplatin.
Example 3: Activity of mer-[RhCl3(DMSO)(pp)] complexes towards lymphoma and leukemia cells
The inhibition of cell proliferation by complexes Rh3a and Rh4a, was also evaluated in vitro in BJAB cells (Burkitt-like lymphoma cells). After an incubation period of 24 h, the viability and cell count were measured with a CASY@CellCounter and Analyser System, with the settings specifically defined for the requirements of the employed cells. The dose dependent decreasing of cell proliferation is depicted for the highly potent rhodium(lll) complexes in Figure 9. IDgg values for these complexes are listed in Table 3. It is apparent that the meridional complexes Rh3a and Rh4a are effective at low micromolecular concentrations in inhibiting cell proliferation for the lymphoma cells
C A
(IDgQ values 0.4 - 0.8 μM). The number of viable cells N are given in units of 10 mL" with values given as the % of control values ± the estimated standard deviation.
Necrotic cell death is characterized by the early release of LDH, whereas apoptotic cells, in contrast, initially retain their membrane integrity and do not exhibit rapid release of large intracellular proteins such as lactate dehydrogenase. Figure 10 depicts the % values of viable cells on the basis of the LDH release established for BJAB cells after 3 h incubation with different concentrations of Rh3a and Rh4a. The highly active rhodium(lll) complexes mer-[RhCI3(DMSO-κS)(pp)] Rh3a and Rh4a for which concentrations in the range 0.4 - 1.0 μM were employed for an incubation period of 3 h have only minor unspecific cytotoxic effects on BJAB cells. These results indicate that necrosis does not have a significant impact on the potency of the complexes Rh3a and Rh4a. Table 3 Biological data for complexes Rh3a and Rh4a in BJAB cells
Dissipation of
Inhibition of
Apoptosis induction mitochondrial membrane
Compound proliferation AC50 (μM)c potentiala'd
ID50 (μM)b c(μM)
Rh3a 0.8 1.2 0.8
Rh4a 0.4 1.0 0.6
50% cells with low Δψm, D after 24 h, c after 75 h, α after 48 h
Apoptosis, in contrast to unspecific necrosis, requires a controlled and regulated mechanism leading to cell death. DNA fragmentation (hypoploidy) is considered to be a typical effect of apoptotic cell death and, therefore, the induction of apoptosis for Rh3a and Rh4a was quantified by flow cytometric measurements of the DNA fragments after incubating lymphoma cells (BJAB) and NALM-6 cells 72 h with the complexes. The numbers of apoptotic NALM-6 cells for different concentrations of Rh3a and Rh4a are illustrated in Figure 1 1 a. This depicts the DNA fragmentation (hypoploidy) after treatment for 72 h. As may be seen, extensive DNA fragmentation is observed even for the very low concentrations (0.3, 0.8 μM) employed for the cytotoxic complexes. The induction of apoptosis in BJAB cells was also confirmed for the meridional trichloridorhodium(lll) complexes (Figure 1 1 b) and this was also the case for the doxorubicin resistant cell line 7CCA when treated with the dpq complex Rh3a but not the dppz complex Rh4a. After incubating BJAB cells with Rh4a for 12h, significant changes could also be identified by fluorescence microscopy.
Acute lymphoblastic leukemia (ALL) is the most common malignant disease in childhood. To determine whether an apoptosis induction can also be found in primary human cells or not, Rh3a and Rh4a were incubated with leukemia cells taken from a patient with relapsed childhood ALL. The isolated primary lymphoblasts were treated with Rh3a and Rh4a at the ID50 concentrations established for BJAB cells and with the cytostatic drugs daunorubicin, doxorubicin and vinchristine. As may be gauged from Figure 12, complexes Rh3a and Rh4a appear to exhibit superior apoptosis induction in comparison to these standard drugs for the treatment of childhood ALL. Fig. 12 depicts the DNA fragmentation after 60 h incubation with Rh3a and Rh4a and the standard cytostatic agents daunorubicin (Daun), doxorubicin (Dox) and Vincristine (Vcr). Values are given in hypoploidy (sub G1 ) which reflects the number of apoptotic cells.
It is clearly demonstrated that complexes Rh3a and Rh4a trigger the mitochondrial pathway of apoptosis. As illustrated in Figure 13, dose dependent loss of the mitochondrial membrane potential was observed for BJAB cells after 48 h of incubation with the meridional rhodium(lll) compounds. After staining the cells with the dye JC-1 (5,5',6,6'-tetrachloro-1 ,1 ,3,3'-tetraethyl-benzimidazolylcarbocyanine iodide), the mitochondrial permeability was quantified by flow cytometric determination of the cells with decreased fluorescence, i.e. with mitochondria displaying a lower membrane potential. Values of the mitochondrial permeability transition are given as percentages of cells with low Δψm.
Cell cultures: BJAB (burkitt-like lymphoma) and NALM-6 (human B cell precursor leukemia) cells were maintained at 37°C in RPMI 1640 (GIBCO, Invitrogen)
A supplemented with 10% heat inactivated fetal calf serum, 100 000 U L penicillin, 0.1
1 1 g-L" streptomycin and 0.56 g-L" L-glutamine. The cells were subcultured every 3-4 days by dilution of the cells to a concentration of 1 • 105 cells mL . Twenty-four hours before the assay setup, cells were cultured at a concentration of 3-105 cells mL to ascertain standardized growth conditions. For apoptosis assays, the cells were then diluted to a concentration of 1 x 105 cells mL immediately before addition of the different complexes. To generate 7CCA (doxorubicin resistant BJAB) cells, BJAB cells
Λ were exposed to an initial concentration of 0.1 μg-L" doxorubicin and then treated with
A doxorubin up to 1 mg-L" , whenever the vitality of the cells was higher than 85%.
Patients: Primary lymphoblasts were obtained by bone marrow aspiration of patients with relapsed acute lymphoblastic leukemia (ALL). The diagnosis was established by immunophenotyping of leukemia cells according to Bene et
Figure imgf000018_0001
* Lymphoblasts and mononuclear cells were separated by centrifugation over Biocoll (Biochrom KG, Berlin, Germany). After separation, the percentage of leukemia cells was above 95%. The leukemia cells were immediately seeded at a density of 3-105 cells mL in RPMI 1640 completed cell culture medium and incubated for 60 h with daunorubicin, doxorubicin and vincristine, as well as with complexes Rh3a and Rh4a at concentrations of their LD^Q values in BJAB cells. The use of the cells is in accordance with the ethical standards of the responsible committee on human experimentation and the Helsinki Declaration as revised in 2000. It is also in accordance with the positive vote of the ethics committee from 14.12.2000 for the ALL-REZ-BFM-study in 2002. Informed signed consent was obtained from either the patient or from their next of kin.
Cytotoxicity measurements: Cytotoxicity of Rh3a and Rh4a towards BJAB cells was measured by release of lactate dehydrogenase (LDH) as described previously. ^ ^ After incubation with different concentrations of the complexes for 1 h or 3 h at 37°C, LDH activity released by BJAB cells was measured in the cell culture supernatants using the Cytotoxicity Detection Kit from Boehringer Mannheim® (Mannheim, Germany). The supernatants were centrifuged at 1500 rpm for 5 min. Cell-free supernatants (20 μL) were diluted with phosphate-buffered saline (PBS, 80 μL) and a reaction mixture containing 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride (INT), sodium lactate, oxidised nicotinamide adenine dinucleotide (NAD+) and diaphorase (100 μL) was added. Time-dependent formation of the reaction product was the quantified photometrically at 490 nm. The maximum amount of LDH activity released by the cells was determined by lysis of the cells by using 0.1% Triton X-100 in culture medium and set as 100% cell death.
Determination of cell viability: Cell viability was determined by using the CASY® Cell Counter + Analyzer System from Innovatis (Bielefeld, Germany). Settings were specifically defined for the requirements of the used cells. With this system, the cell concentration can be analyzed simultaneously in three different size ranges: cell debris, dead cells, and viable cells. Cells were seeded at a density of 1 -105cells/ml and treated with different concentrations of Rh3a and Rh4a; non-treated cells served as controls. After a 24 h incubation period at 37°C, cells were resuspended properly and 100 μl_ of each well was diluted in 10 ml. CASYton (ready-to-use isotonic saline solution) for an immediate automated count of the cells.
Measurement of DNA fragmentation: Apoptotic cell death was determined by a modified cell cycle-analysis, which detects DNA fragmentation at the single cell level as described previously.^ ^ Cells were seeded at a density of 1 -105 cells mL and treated with different concentrations of Rh3a and Rh4a. After a 72 h incubation period at 37°C, cells were collected by centrifugation at 1500 rpm for 5 min, washed with PBS at 40C and fixed in PBS/2% (v/v) formaldehyde on ice for 30 min. After fixation, cells were pelleted, incubated with ethanol/PBS (2:1 , v/v) for 15 min, pelleted and
A resuspended in PBS containing 40 μg mL" RNase A. RNA was digested for 30 min 370C, after which the cells were pelleted once again and finally resuspended in PBS
A containing 50 μg mL" propidium iodide. Nuclear DNA fragmentation was quantified by flow cytometric determination of hypodiploid DNA (Fluorescence - activated cell sort, FACS). Data were collected and analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany) apparatus equipped with the CELL Quest software.
Measurement of the mitochondrial permeability transition: After an incubation period of 48 h with different concentrations of Rh3a and Rh4a, the cells were collected by centrifugation at 1500 rpm, 40C for 5 min. The mitochondrial permeability transition was then determined by staining the cells with 5, 5', 6, 6 '-tetrachloro-1 ,1 ',3,3 '- tetraethyl-benzimidazolylcarbocyanine iodide (JC-1 ; Molecular Probes, Leiden, The Netherlands). 1 - 105 cells were resuspended in 500 μl phenol red-free RPMI 1640 without supplements and JC-1 was added to give a final concentration of 2.5 μg mL" . The cells were incubated for 30 min at 370C with moderate shaking. Control cells were likewise incubated in the absence of JC-1 dye. The cells were harvested by centrifugation at 1500 rpm, 40C for 5 min, washed with ice-cold PBS and resuspended in 200 μl PBS at 40C. Mitochondrial permeability transition was then quantified by flow cytometric determination of the cells with decreased fluorescence, i.e. with mitochondria displaying a lower membrane potential. Data were collected and analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany) apparatus equipped with the CELL Quest software. Data are given in % cells with low ΔΨm, which reflects the number of cells undergoing mitochondrial apoptosis.
Example 4: Synthesis of iridium (III) polypyridyl complexes of the type fac- and mer-lrCI3(DMSO)(pp)
The iridium (III) complexes fac-lrCI3(DMSO)(pp) liia - Ir5a were prepared by stepwise reaction of IrCI3.3H2O with equimolar quantities of the appropriate polypyridyl ligand (pp=bpy, phen, dpq, dppz, dppn) and DMSO in CH3OH solution in the dark. The general procedure is described below for liia.
fac-[lrCI3(DMSO)(bpy)] 2H2O (IrIa)
31.2 mg (0.2 mmol) of 2,2'-bipyridine were added to a solution of 70.5 mg (0.2 mmol) of IrCI33H2O in 10 mL methanol. After stirring at 800C for 2 h in the dark, 0.2 mmol (14.3 μL) DMSO were added and the mixture was heated for a further 2 h at 800C. Following cooling to 25°C and solvent removal under vacuum, the resulting solid was washed with methanol and diethyl ether, and dried in the dark in vacuum. Yield: 70.5 mg (62 %). Anal. Calcd. for Ci2H14CI3lrN2OS-2H2O (M = 568.9): C 25.33, H 3.19, N 4.92: Found C 25.2, H 3.2, N 4.4 %. LSIMS: m/z (%) = 532(18) [M]+, 497(100) [M-HCI] +, 460(27) [M-2HCI]+, 419(23) [M-HCI-DMSO]+. 1H NMR (400 MHz, CD2CI2, 300C in the dark): δ = 3.44 (s, 6H, DMSO-CH3), 7.71 (dd, 2H, H3/H8), 8.23 (dd, 2H, H4/H7), 8.44 (d, 2H, H5/H6), 9.14 (d, 2H, H2/H9) ppm. 1H NMR (400 MHz, D2O, 30°C): δ = 3.50 (s, 6H, DMSO-CH3), 7.56 (dd, 2H, H3/H8), 8.04 (dd, 2H, H4/H7), 8.08 (d, 2H, H5/H6), 8.66 (d, 2H, H2/H9) ppm.
fac-[lrCI3(DMSO)(phen)] CH3OH H2O (Ir2a)
Preparation as for IrIa with 36.0 mg (0.2 mmol) of 1 ,10-phenanthroline. Yield: 76.5 mg (63 %). Anal. Calcd. for Ci4H14CI3IrN2OS CH3OH H2O (M = 607.0): C 29.68, H 3.32, N 4.62: Found C 29.7, H 3.3, N 4.5 %. LSIMS: m/z (%) = 556(9) [M]+, 521 (30) [M-HCI]+, 443(15) [M-HCI-DMSO]+. 1H NMR (400 MHz, CD2CI2, 30°C in the dark): δ = 3.50 (s, 6H, DMSO-CH3), 8.01 (dd, 2H, H3/H8), 8.07 (s, 2H, H5/H6), 8.66 (d, 2H, H4/H7), 9.64 (d, 2H, H2/H9) ppm. 1H NMR (400 MHz, D2O, 300C): δ = 3.51 (s, 6H, DMSO-CH3), 7.81 (dd, 2H, H3/H8), 7.95 (s, 2H, H5/H6), 8.48 (d, 2H, H4/H7), 9.05 (d, 2H, H2/H9) ppm. Crystals of mer-[lrCI3(DMSO)(phen)] Ir2b were grown by slow evaporation of an H2O/CH3OH solution. 1H NMR (400 MHz, CD2CI2, 300C, in the dark): δ = 3.68(s, 6H, DMSO-CH3), 7.89, 8.05 (2dd, 2H, H3/H8), 8.07 (2d, 2H, H5/H6), 8.44, 8.57 (2d, 2H, H4/H7), 9.98 (d, 1 H, H2), 10.29 (d, 1 H, H9) ppm.
fac-[lrCI3(DMSO)(dpq)] 3H2O (Ir3a)
Preparation as for Ilia with 46.4 mg (0.2 mmol) of dipyrido[3,2-f:2',3'-/?]quinoxaline. Yield: 87.5 mg (66 %). Anal. Calcd. for Ci6H14CI3IrN4OS SH2O (M = 663.0): C 28.99, H 3.04, N 8.45: Found C 28.8, H 3.1 , N 8.5 %. LSIMS: m/z {%) = 610(9) [M]+, 573(35) [M- HCI]+, 495(29) [M-HCI-DMSO]+. 1H NMR (400 MHz, CD2CI2, 300C in the dark): δ = 3.49 (s, 6H, DMSO-CH3), 7.85 (dd, 2H, H3/H8), 9.01 (s, 2H, H1 1/H12), 9.33 (d, 2H, H4/H7), 9.55 (d, 2H, H2/H9) ppm. 1H NMR (400 MHz, CD3OD, 30°C): δ = 3.49 (s, 6H, DMSO- CH3), 8.14 (dd, 2H, H3/H8), 9.17 (s, 2H, H1 1/H12), 9.28 (d, 2H, H4/H7), 9.78 (d, 2H, H2/H9) ppm.
fac-[lrCI3(DMSO)(dppz)]2H2O (lr4a)
Preparation as for IrIa with 56.4 mg (0.2 mmol) of dipyrido[3,2-a:2',3'-c]phenanzine. Yield: 94.5 mg (68 %). Anal. Calcd. for C20H16CI3IrN4OS 2H2O (M = 695.0): C 34.56, H 2.90, N 8.06: Found C 34.4, H 3.2, N 7.8 %. LSIMS: m/z (%) = 625(6) [M-HCI]+, 587(13) [M-2HCI]+. 1H NMR (400 MHz, CD2CI2, 300C in the dark): δ = 3.51 (s, 6H, DMSO-CH3), 8.06 (dd, 2H, H12/H13), 8.19 (dd, 2H, H3/H8), 8.42 (dd, 2H, H1 1/H14), 9.72 (d, 2H, H4/H7), 9.95 (d, 2H, H2/H9) ppm. 1H NMR (400 MHz, CD3OD, 30°C): δ = 3.46 (s, 6H, DMSO-CH3), 8.15 (dd, 2H, H12/H13), 8.31 (dd, 2H, H3/H8), 8.50 (dd, 2H, H1 1/14), 9.31 (d, 2H, H4/H7) 10.02 (d, 2H, H2/H9) ppm.
fac-[lrCI3(DMSO)(dppn)] 2CH3OH (Ir5a)
Preparation as for Ilia with 66.4 mg (0.2 mmol) of benzo[i]dipyrido[3,2-a:2',3'- φhenanzine. Yield: 108.0 mg (70 %). Anal. Calcd. for C24H18CI3IrN4OS 2CH3OH (M = 773.2): C 40.39, H 3.39, N 7.25: Found C 40.8, H 3.4, N 7.3 %. LSIMS: m/z (%) = 674(5) [M-HCI]+, 595(3) [M-HCI-DMSO]+. 1H NMR (400 MHz, CD2CI2, 30°C in the dark): δ = 3.39 (s, 6H, DMSO-CH3), 7.62 (dd, 2H, H13/H14), 7.80 (dd, 2H, H3/H8), 8.21 (dd, 2H, H12/H15), 8.98 (s, 2H, H1 1/H16), 9.22 (d, 2H, H4/H7), 9.67 (d, H2/H9) ppm. 1H NMR (400 MHz, [D6]DMSO, 300C): δ = 3.33 (s, 6H, DMSO-CH3), 7.73 (d, 2H, H13/H14), 8.25 (dd, 2H, H3/H8), 8.36 (d, 2H, H12/15), 9.12 (s, 2H, H11/H16), 9.29 (d, 2H, H4/H7), 9.76 (d, 2H, H2/H9) ppm.
X-ray structural analysis of mer-[lrCI 3 (DMSO)(phen)] Ir2b (Fig. 7) Crystals of Ir2b suitable for X-ray analyses were grown over a period of 7 days by slow evaporation of a solution of Ir2a in water/methanol. Ci4H14CIsIrN2OS, M = 556.9, triclinic, space group PΪ (no. 2), a = 9.069(2), b = 1 1.989(2), c = 16.958(3) A, α = 91.89(3), β = 103.92(3), γ = 92.68(3)°, V = 1785.8(6) A3, Z = A, D^ = 2.071 g cm"3, μ(MoKα) = 8.043 mm"1; Siemens P4 diffracto meter using graphite-monochromated MoK radiation (λ = 0.71073 A). Intensity data were collected using ω scans in the range 4° < 2Θ < 50° and the data were corrected semi-empirically for absorption using ω scan data. The structure was solved by direct methods with SHELX97 and refined
2 against F using SHELXL97 (G. M. Sheldrick, Gottingen, Germany, 1997). Anisotropic temperature factors were employed for non-hydrogen atoms and protons were included at geometrically calculated positions as riding atoms. The final R factors were Ri =
2 2 2 2 1/2
0.086 for 2678 reflections with I > 2 σ(l) and wR2 = {[ΣW(FQ -F0 ) /Σw(FQ) ]} = 0.240, S
(goodness-of-fit) = 0.915 for all 6087 independent reflections.
Electronic spectra and DNA binding studies UV/vis spectra were recorded with an Analytik Jena SPECORD 20 spectrometer and CD spectra with a Jasco J-715 instrument in the range 220 - 500 nm for 1 :10 complex/[DNA] mixtures [complex = 20 μM, DNA concentration in M(Nucleotide) = 200 μM] in a 10 mM phosphate buffer at pH 7.2. To assure solubility of the complexes, 1 % DMSO was added to all of the aqueous buffer solutions. Thermal denaturation temperatures Tm of the 1 :10 complex/[DNA] mixtures were measured by recording melting curves at 1 °C steps for the wavelength 260 nm on the SPECORD 200 spectrometer equipped with a Peltier temperature controller. Tm values were calculated by determining the midpoints for melting curves from first-order derivatives and are estimated to be accurate within ± 1 °C. Concentrations of CT DNA were determined spectrophotometrically using the molar extinction coefficient ε = 6600 M"1 cm"1. Mass spectrometry and NMR spectroscopy
LSIMS spectra (LSIMS = liquid secondary ion mass spectrometry) were registered for the mass range m/z < 3000 with a Fisons VG Autospec employing a caesium ion gun (voltage 17 kV) and 3-nitrobenyzl alcohol as the liquid matrix. A Bruker DRX 400 was employed to record 1H and 13C NMR spectra with chemical shifts reported as δ values relative to the signal of the deuterated solvent.
The fac isomers of liia - Ir5a are stable in light-protected CD2CI2 solution but, with the exception of Ir5a, isomerize rapidly to a mixture of the fac and mer isomers in the presence of light. In contrast, solutions of the fac isomers in the polar solvents D2O and
CD3OD are stable under such conditions. The isomer mer-[lrCl3(DMSO-κS)(phen)]
Ir2b was, however, isolated by slow evaporation of an H2O/CH3OH solution of Ir2a and characterized by X-ray structural analysis. UVA/is and CD studies of the interaction of IrIa - Ir5a with calf thymus DNA are in accordance with an absence of intercalation
A and H NMR studies indicate that the complexes do not react with the guanine N7 atom of 5'-GMP2'.
The structures of the fac complexes liia - Ir5a and of the mer complexes liib - Ir3b are shown in Figures 5 and 6.
Example 5: Cytotoxic Measurements and cellular uptake studies fac-
IrCI3(DMSO)(Pp) complexes and cellular uptake
The antiproliferative effects of the compounds IrIa - Ir5a were determined as described in Example 2.
For cellular uptake studies, HT-29 and MCF-7 cells were grown until at least 70 %
2 confluency in 175 cm cell culture flasks [16]. Stock solutions of complexes IrI a - Ir5a in DMSO were freshly prepared and diluted with cell culture medium to the desired concentrations (final DMSO concentrations: 0.1 % v/v, final complex concentration: 10 or 100 μM). The cell culture medium of the cell culture flasks was replaced with 1 O mL of the cell culture medium solutions containing IrIa - Ir5a (10 μM or 100 μM) and the flasks were incubated for 6 h at 37°C/5 % CO2. The cell pellets were isolated, resuspended in 1 - 5 ml. twice suspended water, lysed by using a sonotrode and approximately diluted using twice distilled water. The iridium content of the samples was determined by graphite furnace atomic absorption spectroscopy (GF-AAS) and the protein content of separate aliquots by the Bradford method. To correct for matrix effects in GF-AAS measurements, samples and standards were adjusted to the same protein concentration by dilution with twice distilled water. Prior to GF-AAS analysis 20 μl_ triton X-100 (1 %) and 20 μl_ hydrochloric acid were added to each 200 μl_ sample of the cell suspensions. Cellular uptake was expressed as ng iridium per mg cell protein for data obtained from 3 independent experiments. Conversion of the ng iridium/mg protein value to the micromolar cellular concentration was performed as described previously [16].
Atomic Absorption Spectroscopy
A Vario 6 graphite furnace atomic absorption spectrometer (Analytik Jena) was employed for the Ir quantification using a wavelength of 208.9 nm with a bandpass of
0.5 nm. A deuterium lamp was used for background correction. Matrix containing standards were obtained by addition of an iridium stock solution (1 mg/mL Ir in 5 %
HCI). 18 % hydrochloric acid solution was employed as a modifier [16]. Probes were injected at a volume of 25 μl_ into graphite tubes. Drying, pyrolysis and atomization in the graphite furnace was performed according to the conditions listed in Table 1. The detection limit for the method was 3.8 μg Ir L . The mean AUC (area under curve) absorptions of duplicate injections were used throughout the study.
The IC50 values and cellular uptake results are summarized in Tab. 2.
Table 2 IC5Qvalues (μM) and cellular uptake (ng Ir/mg protein) of the complexes fac-[lrCI3(DMSO)(pp)] IrI a - Ir5a in comparison to other relevant compounds
MCF-7 uptake HT-29 uptake
Cpd. PP MCF-7 IC50 HT-29 IC50 10 μM 100 μM 10 μM 100 μM
IrIa bpy > 100 > 100 n.d. n.d. 0.0 0.0
Ir2a phen 4.6(0.5) 4.6(0.2) 0.0 74.4(13.5) 0.0 21.2(5.2)
Ir3a dpq 5.5(0.9) 6.1 (0.7) 0.3(0.3) 640.5(148.9) 0.0 167.8(3.4)
Ir4a dppz 0.8(0.3) 1.5(0.2) 3.3(3.3) 644.0(149.1 ) 1 1.9(3.0) 1664.4(473.1 )
Ir5a dppn 0.21 (0.1 1 ) 1.3(0.4) 19.3(0.8) 180.8(2.7) 37.4(8.9) 226.1 (26.6) κ>
Ul 6 dppz 2.3(0.4) 7.4(0.9)[15] n.d. n.d. 70.4(1.0) n.d
7 dppn 0.17(0.02) 0.41 (0.16)[15] n.d. n.d. 149.6(7.8) n.d dppz 0.8(0.6) 1.8(0.2) n.d. n.d. n.d. n.d. cisplatin 2.0(0.3) 7.0(2.0) n.d. n.d. n.d. n.d. n.d.: not determined
0.0: value below the detection limit
6 = [(η5-C5Me5)lrCI(dppz)](CF3S03) [5]
7 = [(T^-C5Me5)IrUMe2N)2CSXdPPn)](CF3SO3) [5]
The cytotoxity results are also depicted in Fig. 8 and indicate that, with the exception of the bpy complex Ilia, the compounds of the typ fac-lrCI3(DMSO)(pp) are potent cytotoxic agents, in particular the dppz and dppn complexes Ir4a and Ir5a.
In the following, the invention will be explained in more detail with reference to Figures 1 - 13.
Figure Captions
Figure 1 : Structures of the trichloridorhodium(lll) polypyridyl complexes mer- [RhCI3(DMSO-KS)(Pp)] RMa - Rh5a (pp = bpy, phen, dpq, dppz, dppn)
Figure 2: Structures of the fac isomers fac-[RhCI3(DMSO-κS)(pp)] RhI b (pp = bpy) and Rh2b (pp = phen)
Figure 3: Molecular structure of fac-[RhCI3(DMSO-κS)(bpy)] Rh1 b Figure 4: IC50 values for complexes RMa - Rh5a against the human cell lines MCF- 7 (breast cancer) and HT-29 (colon cancer)
Figure 5: Structures of the fac complexes Ilia - Ir5a
Figure 6: Structures of the mer complexes Ir1 b - Ir3b
Figure 7: Molecular structure of mer-[lrCl3(DMSO)(pp)] Ir2b as established by X-ray structural analysis
Figure 8: IC5O values for the complexes Ir2a - Ir5a against the human cell lines MCF-7 (breast cancer) and HT-29 (colon cancer)
Figure 9: Inhibition of cell proliferation in Burkitt like lymphoma cells after treatment
(R) with complexes Rh3a and Rh4a for 24 h as measured by a CASY cell counter (control = untreated cells). N = number of cells in units of
10 • ml. with values given as the % of control values ± esd (n = 3), / = percentage inhibition of cell proliferation. 1 -10 BJAB cells normally grow
C A up to 2.5-10 cells ml_~ in 24 h in the absence of proliferation inhibitors.
Figure 10: BJAB cell viability values for complexes Rh3a and Rh4a. The cell viability was determined using the LDH release assay after an incubation period of 1 h. Figure 11 : Apoptosis induction as measured by DNA DNA fragmentation in (a) leukemia cells (NALM-6) and (b) lymphoma cells (regular BJAB and doxorubicin resistant BJAB cells = Doxo7CCA) after treatment for 72 h with different concentrations of Rh3a, Rh4a and doxorubicin (Dox). Data are given in % hypoploidty (subG1 ) ± esd (n = 3), which reflects the number of apoptotic cells.
Figure 12: Apoptosis induction as measured by DNA fragmentation in primary leukemia cells isolated from a patient with relapsed childhood ALL after treatment for 60 h with Rh3a, Rh4a and standard cytostatic agents of clinical use (Daun = daunorubicin, Dox = doxorubicin, Vcr = vincristine).
All cytostatic agents were applied at the LC50 concentrations determined for BJAB cells. Data are given in % hypoploidty (subG1 ) ± esd (n = 3), which reflects the number of apoptotic cells.
Figure 13: Mitochondrial permeability transition as measured by flow cytometric analysis in lymphoma cells (BJAB) after treatment with different concentrations of (a) Rh3a and Rh4a for 48 h. Values of the mitochondrial permeability transition are given as percentages of cells with low Δψm ± esd (n = 3).
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[16] Ott, I. et al., ChemMedChem., 2007, 702-707
[17] Bene, M. C. et al., Leukemia 1995, 9, 1783 - 1786 [18] Diller, R. A. et al., Chem. Biodiversity 2005, 2, 1331 - 1337
[19] Wieder, T. et al., Leukemia 2001 , 15, 1735 - 1742.

Claims

Claims
1. A pharmaceutical composition comprising as active substance one or more octahedral metal (III) polypyridyl complexes of general formula I
Me(hal)3(sol)(pp) (I)
where
Me represents rhodium or iridium hal is a halogenide selected from the group consisting of chlorine, bromine, fluorine and iodine or a pseudohalogenide selected from the group consisting of SCN, NCO or N3, sol is a solvent selected from the group consisting of DMSO, H2O, CH3OH, DMF and CH3CN and pp is a polypyridyl ligand selected from the group constisting of 2,2'-bipyridine (bpy), 1 ,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz) and benzo[i]dipyrido[3,2-a:2',3'- c]phenazine (dppn), optionally substituted with one or more of the substituents selected from the group constisting of hydroxy, -COOR, -SO3H, -CHO, -CH3, - CF3, -OCH3, -OC2H5, -NO2, -CN, -NH2, phenyl and halogenide, wherein R = H, - CH3 Or -C2H5,
and their physiologically tolerated isomers, and hydrates, solvates or salts thereof.
2. The pharmaceutical composition of claim 1 , wherein sol in formula (I) represents DMSO or H2O.
3. The pharmaceutical composition of claim 1 or 2, wherein hal in formula (I) represents chlorine or bromine.
4. The pharmaceutical composition of anyone of claims 1 to 3, wherein the polypyridyl ligand in formula (I) represents phen, dpq, dppz or dppn, preferably dpq, dppz or dppn.
5. The pharmaceutical composition of anyone of claims 1 to 4, wherein the complexes of formula (I) are hydrates or solvates.
6. The pharmaceutical composition of anyone of claims 1 to 5, wherein the complexes of formula (I) are [Me(hal)3(sol)(pp)](CH3OH)m (H2O)n, wherein m is 0; 1 or 2 and n is 0; 1 ; 1 ,5; 2 or 3.
7. The pharmaceutical composition of anyone of claims 1 to 6, wherein the complexes of formula (I) are the fac- or mer-isomers.
8. The pharmaceutical composition of anyone of claims 1 to 7, wherein the complexes of formula (I) are mer-RhCI3(DMSO)(pp), fac-RhCI3(DMSO)(pp), mer-RhCI3(DMSO-H2O)(pp), fac-RhCI3(DMSO-H2O)(pp), fac-RhCI3(H2O)(pp), mer-RhCI3(H2O)(pp), fac-lrCI3(DMSO)(pp), mer-lrCI3(DMSO)(pp), fac-lrCI3(H2O)(pp), mer-lrCI3(H2O)(pp), fac-lrBr3(H2O)(pp), mer-lrBr3(H2O)(pp), and their hydrates or solvates.
9. Octahedral metal (III) polypyridyl complexes of general formula (I) according to claims 1 to 8 for prevention and treatment of cancer and its metastases, preferably breast, colon and colorectal cancer, lymphoma and leukaemia.
10. Use of one or more octahedral metal (III) polypyridyl complexes of general formula (I) according to claims 1 to 8 for the manufacture of a pharmaceutical composition for prevention and treatment of cancer and its metastases, preferably breast, colon and colorectal cancer, lymphoma and leukaemia.
1 1. Octahedral metal (III) polypyridyl complexes of general formula Ib Me(hal)3(sol)(pp) (Ib)
where
Me represents rhodium or iridium hal is a halogenide selected from the group consisting of chlorine, bromine, fluorine and iodine or a pseudohalogenide selected from the group consisting of SCN,
NCO or N3, sol is a solvent selected from the group consisting of DMSO, DMF and CH3CN and pp is a polypyridyl ligand selected from the group constisting of 2,2'-bipyridine
(bpy), 1 ,10-phenanthroline (phen), dipyrido[3,2-f:2',3'-h]quinoxaline (dpq), dipyrido[3,2-a:2',3'-c]phenazine (dppz) and benzo[i]dipyrido[3,2-a:2',3'- c]phenazine (dppn), optionally substituted with one or more of the substituents selected from the group constisting of hydroxy, -COOR, -SO3H, -CHO, -CH3, -
CF3, -OCH3, -OC2H5, -NO2, -CN, -NH2, phenyl and halogenide, wherein R = H, -
CH3 Or -C2H5,
and their physiologically tolerated isomers, and hydrates, solvates or salts thereof.
12. Complexes of claim 1 1 , wherein sol represents DMSO.
13. Complexes of claim 1 1 or 12, wherein hal represents chlorine or bromine.
14. Complexes of anyone of claims 1 1 to 13, wherein the polypyridyl ligand represents phen, dpq, dppz or dppn, preferably dpq, dppz or dppn.
15. Complexes of anyone of claims 1 1 to 14 being hydrates or solvates.
16. Complexes of anyone of claims 1 1 to 15 being [Me(hal)3(sol)(pp)](CH3OH)m (H2O)n, wherein m is O; 1 or 2 and n is 0; 1 ; 1 ,5; 2 or 3.
17. Complexes of anyone of claims 1 1 to 16 being the fac- or mer-isomers.
18. Complexes of anyone of claims 11 to 17, being mer-RhCI3(DMSO)(pp)! fac-RhCI3(DMSO)(pp)! mer-RhCI3(DMSO-H2O)(pp), fac-RhCI3(DMSO-H2O)(pp), fac-lrCI3(DMSO)(pp), mer-lrCI3(DMSO)(pp)! and their hydrates or solvates.
19. A method for treating cancer which comprises administering to a patient suffering from cancer a therapeutically effective amount of one or more octahedral metal (III) polypyridyl complexes of general formula (I) according to claims 1 to 8.
PCT/EP2008/067021 2007-12-20 2008-12-08 Pharmaceutical compositions comprising octahedral metal (iii) polypyridyl complexes and their use in prevention and treatment of cancer WO2009080482A1 (en)

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WO2004012667A2 (en) * 2002-08-01 2004-02-12 Purdue Research Foundation Photoactivated anti-viral and anti-cancer agent

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